The continuous increase of pathogenic bacteria resistant to the existing antibiotics is a health global concern, and so there is a pressing need to discover and develop new compound that are active against resistant bacteria. This has led to renewed interest in natural products which have been in the past a rich source of antibiotics such as penicillins, macrolides and glycopeptides. Attractive candidates are also antimicrobial peptides and among them those designated as lantibiotics, i.e. lanthionine-containing antibiotic. Lantibiotics form a particular group within the antimicrobial peptides and are distinguished by several features such as primary and spatial structure characteristics, unique biosynthetic pathways and peptide modification reactions and potent antibacterial activity. These are a group of peptide-derived antimicrobial compounds secreted by Gram-positive bacteria and primarily act on Gram-positive bacteria. Lantibiotics are ribosomally synthesised as prepropeptides which are posttranslationally modified to their biologically active forms. The prepeptide consists of an N-terminal leader sequence, that does not undergo any post-translational modification and is cleaved off during or after secretion from the cell, and a C-terminal region (the propeptide), which is post-translationally modified. Lantibiotics are produced by different bacteria: the common feature of these compounds is the presence of one or more lanthionine residues, which consist of two alanine residues covalently cross-linked by a thioether linkage The thioether brigde is formed when a cysteine residue reacts with a dehydroalanine or dehydrobutyrine moiety to form a lanthionine or methyllantionine residue, respectively. The dehydroamino acid residues are in turn formed by dehydration of serine and threonine, respectively. Based on their structural and functional properties, lantibiotics are usually divided in two groups, type-A and type-B. Type-A lantibiotics are elongated, cationic peptides varying in length from 20 to 34 amino acids residues: raisin, subtilin, epidermin and Pep5 are members of this group. Type-B are globular peptides with a net negative charge: examples of this group of lantibiotics are mersacidin, cinnamycin, lacticin 481 and actagardine. These structural differences reflect on the mechanism of action. Type-A compounds exert their antimicrobial activity by blocking cell wall biosynthesis and by forming pores in the cellular membranes, through a mechanism that may or may not be aided by prior docking on the cellular target lipid II. Type-B lantibiotics also exert their antimicrobial activity by inhibiting peptidoglycan biosynthesis, but these compounds do not form pores once bound to lipid II.
Lantibiotics have been shown to have efficacy and utility as food additives and antibacterial agents. Nisin, the most studied lantibiotic, is produced by Lactococcus lactis and is active at low concentrations (low nanomolar MICs) against many Gram-positive bacteria including drug resistant strains and the food-borne pathogens Clostridium botulinum and Listeria monocytogenes. It has been extensively used as a food preservative without substantial development of bacterial resistance. Other lantibiotics show interesting biological activities: for example, epidermin shows high potency against Propionibacterium acnes; cinnamycin and duramycin inhibit phospholipase A2 and angiotensin converting enzyme, providing potential applications as anti-inflammatory agents and for blood pressure regulation, respectively; and mersacidin inhibits many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA).
The genes responsible for the biosynthesis of lantibiotics are organized in clusters designated by the locus symbol Ian, with a more specific genotypic designation for each lantibiotic (e.g., nis for nisin, gdin for gallidermin, cin for cinnamycin). Many Ian genes have been sequenced demonstrating a high level of similarity in gene organization. Each cluster includes: the structural gene lanA encoding the prepeptide; and the gene(s) required for the dehydration of Ser and Thr residues in the propeptide portion of LanA and for the thioether formation. For Type-A lantibiotics, LanB carries out the dehydration reactions and LanC is devoted to thioether bridging, whereas in Type-B lantibiotics a single LanM enzyme catalyzes both reactions. Additional genes are usually present in lantibiotic clusters: lanT encodes the ABC transporter for secretion of the lantibiotic, often in combination with a second transport system, encoded by the lanEFG genes; lanP encodes the processing protease, but some clusters lack such a gene which may be part of lanT or its function may be provided by a cellular protease; and lanI encodes a protein involved in self-protection; and lanKR are responsible for regulating expression of the lan genes.
There exists the potential and the utility to obtain improved lantibiotics by manipulation of occurring natural compounds. However, lantibiotics are structurally complex peptides and their accessibility to chemistry is limited to a few positions in the molecule. One of the major limitations for chemistry is to change the type or order of amino acids present in the peptide backbone. In light of the above, it would be desirable to have genes and enzymes useful for redirecting these steps in lantibiotic formation, in order to obtain derivatives that are hard or impossible to make by chemical means. General methods for the design of novel lantibiotic derivatives directly by fermentation processes with precisely engineered strains would thus be highly desirable.
In fact unusual aminoacids in lantibiotics solely contribute to their biological activity and also enhance their structural stability. Enzymes involved in lantibiotic biosynthesis represent a high potential for peptide engineering by introducing unusual aminoacids into desired peptides.
The lantibiotic 107891 shows antibacterial activity against Gram-positive bacteria including methycillin- and vancomycin-resistant strains but shows limited activity against Gram-negative bacteria (for examples, some Moraxella, Neisseria and Haemophilus spp.). 107891 was isolated from fermentation of Microbispora sp. PTA-5024 (WO 2005/04628 A1). It consists of a complex of closely related factors A1 and A2, whose structure can be reconducted to a peptide skeleton, 24 amino acids long, containing lanthionine and methyllanthionine as constituents. In addition, a chlorine atom and one or two —OH residues are present on the molecule. The structure of the components of the 107891 complex is represented by the formula 1 of FIG. 3, where R represents [OH] with the factors A1(R═OH), factor A2 (R═—(OH)2). 107891 appears to combine elements of Type-A and -B lantibiotics: rings A and B in 107891 are highly related to the equivalent rings in Type-A compounds; however, as in Type-B lantibiotics, 107891 is rather globular, it lacks a flexible C-terminal tails and is devoid of charged amino acid residues. Consequently, it cannot be predicted whether the Zan cluster devoted to 107891 formation would encode a single LanM enzyme or separate LanB and LanC proteins. Furthermore, there are no precedent for chlorine-containing lantibiotics, thus the genes responsible for this post-translational modification cannot be predicted from available data.
The design of industrial processes for antibiotic production has been relatively successful, resulting in large size fermentations with antibiotic titers reaching levels of several grams per liter. This has been achieved largely by following empirical, trial and error approaches, and lacks a rational basis. Development of new processes and improvement of current technology thus remains time consuming and may result in bacterial cultures that are unstable, perform inconsistently and accumulate unwanted by-products. In recent years, rational methods have been applied successfully to increase the level of antibiotic produced by Streptomyces spp., which have often involved the manipulation of key regulatory elements present within the gene cluster of interest or the overexpression of rate-limiting steps in the pathway. Therefore, the genes encoding such cluster-associated regulators or limiting steps in the synthesis can be effective tools for yield improvement. However, the cluster-associated regulators so far identified in actinomycetes belong to several different protein families. Even within one family, there is considerable variation in sequence identity. Therefore, the existence, nature, number and sequence of cluster-associated regulators cannot be predicted by comparison to other cluster, even those specifying a related antibiotic. As an example, the tylosin gene cluster encodes four distinct regulators, while none has been found in the cluster specifying the related macrolide antibiotic erythromycin. Similarly, the nature and reason for a rate-limiting step in a biosynthetic pathway cannot be established a priori.
Therefore, tools for increasing the 107891 yield would be highly desirable. However, there are no examples of clusters from other members of the genus Microbispora. Therefore, the mechanism(s) cannot be predicted through which the producer strain protects itself from the action of 107891, governs the expression of the other lan genes, or coordinates expression of lan genes with its other cellular processes. Information about these will be very be useful for optimizing the production process.